Proteinaceous adjuvants

ABSTRACT

A modulated immune response to an antigen is achieved by coadministering the antigen and a genetically-detoxified  pertussis  holotoxin, particularly one retaining its immunogenicity, to a host. The modulated immune response enables immunogenic compositions, including multivalent pediatric vaccines such as DTP, to be provided which produce a modulated immune response in the absence of extrinsic adjuvants such as alum. The adjuvanting effect achieved by the genetically-detoxified  pertussis  holotoxin enables at least the same level of adjuvanting effect to be achieved as previously attained by alum, without the undesirable side effects thereof.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending U.S. patentapplication Ser. No. 08/258,228 filed Jun. 10, 1994.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with proteinaceous adjuvants., i.e. materialswhich modulate immune responses to an antigen.

BACKGROUND OF THE INVENTION

Vaccines have been used for many years to protect humans and animalsagainst a wide variety of infectious diseases. Such conventionalvaccines consist of attenuated pathogens (for example, polio virus),killed pathogens (for example, Bordetella pertussis) or immunogeniccomponents of the pathogen (for example, diphtheria toxoid). Someantigens are highly immunogenic and are capable alone of elicitingimmune responses. Other antigens, however, fail to induce, for example,a protective immune response or induce only a weak immune response.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants. Adjuvants enhance the immunogenicity ofan antigen but are not necessarily immunogenic themselves.

Immunostimulatory agents or extrinsic adjuvants have been used for manyyears to improve the host immune responses to immunogenic compositionsincluding vaccines. Extrinsic adjuvants are immunomodulators which aretypically non-covalently linked to antigens and are formulated toenhance the host immune responses. Thus, adjuvants have been identifiedthat enhance the immune response to antigens delivered parenterally.Some of these adjuvants are toxic, however, and can cause undesirableside-effects, making them unsuitable for use in humans and many animals.Indeed, only aluminum hydroxide and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants in humanand veterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established and,more recently, a HBsAg vaccine has been adjuvanted with alum. While theusefulness of alum is well established for some applications, it haslimitations. For example, it is ineffective for influenza vaccinationand inconsistently elicits a cell mediated immune response. Theantibodies elicited by alum-adjuvanted antigens are mainly of the IgG1isotype in the mouse, which may not be optimal for protection by somevaccinal agents.

Furthermore, studies in rats have demonstrated that alum acts as an IgEadjuvant (ref. 1—Throughout this application, various references arereferred to in parenthesis to more fully describe the state of the artto which the invention pertains. Full bibliographic information for eachcitation is found at the end of the specification, immediately precedingthe Claims. The disclosure of other references are incorporated byreference into the present disclosures. Studies with tetanus anddiphtheria toxoid vaccines also indicate that alum adsorption ofvaccines induces IgE antibodies in humans (refs. 2, 3, 4). Therefore,although the inclusion of an aluminum salt in a vaccine formulation mayimprove its immunogenicity and potency, the fact that it can inducelocal granulomas and IgE antibodies which may contribute tohypersensitivity reactions warrants careful examination of the practiceof alum-adsorption of vaccines for human and animal use.

Some characteristics of desirable adjuvants include:

(1) a lack of toxicity;

(2) an ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) an ability to elicit both cellular and humoral immune responses toantigens administered by various routes, if required;

(5) synergy with other adjuvants;

(6) a capability of selectively interacting with populations of antigenpresenting cells (APC);

(7) an ability to elicit appropriate T_(H)1 or T_(H)2 cell-specificimmune responses;

(8) an ability to selectively increase appropriate antibody isotypelevels (for example, IgA) against antigens; and

(9) that they do not contribute to hypersensitivity reactions.

Of relevance to the present invention is a discussion of the developmentof pertussis vaccines presented below.

Thus, pertussis or whooping cough is a serious respiratory diseasecaused by the infection of the respiratory tract by the gram negativeorganism Bordetella pertussis. Pertussis is a major cause of childhoodmorbidity and is implicated in 360,000 deaths annually (ref. 5). Themost effective method of control of the spread of the disease has provento be the use of widespread immunization programs. The whole cellpertussis vaccine which was shown to have clinical efficacy in the1950's, has been effective in controlling pertussis epidemics (refs. 6,7, 8). The value of the vaccine was illustrated when Japan, Sweden andGreat Britain abandoned routine childhood pertussis immunization.Shortly thereafter, these countries experienced major epidemics ofpertussis (refs. 9, 10, 11, 12).

Although the whole cell pertussis vaccine is effective in preventing theincidence and spread of disease, the acceptance and uptak of the vaccinehas been limited due to reports of vaccine associated adverse effects.Therefore, an impetus for the creation of a non-reactogenic, effectiveand well defined acellular component pertussis vaccine was created. Oneof the key features of the acellular vaccine is the chemicallydetoxified pertussis toxin (PT) component. The presence of nativepertussis toxin in the whole cell vaccine has been a source of concernas studies in animal models have shown that it can induce lymphocytosis,histamine sensitization, potentiation of anaphylaxis and IgE antibodies,enhancement of insulin secretion and many other systemic effects (ref.13). The acellular pertussis vaccines differ with respect to thecombinations and quantities of Bordetella pertussis antigens included inthe vaccines but the key antigens include the agglutinogens, pertactin,filamentous hemagglutinin (FHA) and pertussis toxin (PT). Although theacellular vaccine has been demonstrated to be immunogenic and ofcomparable efficacy to the whole cell vaccine, it has not been aseffective in preventing bacterial colonization (ref. 14). In addition,the results from a Swedish field trial comparing acellular and wholecell pertussis vaccines indicated that the formaldehyde inactivatedpertussis toxin present in the acellular vaccines showed evidence ofreversion to toxicity (ref. 15). Therefore, other methods ofinactivating the pertussis toxin molecule were required.

To overcome the drawbacks of chemical detoxification, several groupsdeveloped genetically detoxified pertussis mutant holotoxin molecules(refs. 16, 17, 18, 19, 20, 21). A promising candidate was the K9G129mutant. Not only was the immunogenicity of the molecule retained, butthe toxicity of this recombinant toxin was greatly diminished (refs. 18,19, 21). In addition, immunization with the K9G129 mutant stimulatedboth humoral and cellular pertussis antigen specific responses (ref.22). Although many clinical trials base the evaluation of theimmunogenicity of a vaccine solely on the antibody response followingimmunization, studies indicate an important role for cellular immunityin protection against this disease. In animal models, the cellularimmune response has been demonstrated to be important in the protectiveresponse against pertussis as the adoptive transfer of cells fromconvalescent animals into sublethally irradiated animals conferredprotection from challenge with Bordetella pertussis organisms while thepassive transfer of immune serum did not (refs. 23, 24). A retrospectivestudy in humans indicated that cell mediated immunity to Bordetellapertussis correlated with a positive history of pertussis (ref. 25).Following natural pertussis infection in humans, both an antibody andcellular immune response are observed (ref. 26). However, immunizationwith either the whole cell or acellular component vaccines resulted invariable pertussis antigen-specific cellular immune responses (refs. 27,28). It appeared that the chemical detoxification of the pertussis toxincomponent destroyed its T cell immunogenicity while the antibodyresponses were unaffected (ref. 26). Therefore, only the geneticallydetoxified pertussis toxin molecule could be used to stimulate both acellular and humoral immune response.

The use of the recombinant PT mutant, K9G129, as a pertussis vaccinecomponent has been well described. A number of different forms of thevaccine have been suggested. Two formulations have been evaluated inhumans. The first formulation consisted of 15 μg of the PT mutant whichwas alum-adsorbed with a total of 0.5 mg of alum per dose (refs. 22, 29)while the other formulation contained 7.5 μg of the K9G129 mutant aswell as 10 μg FHA and 10 μg pertactin and was also alum adsorbed (ref.30). These studies indicated that the genetically detoxified pertussisvaccine candidate was not only safe, immunogenic and could induce a cellmediated response, but, when combined with the FHA and pertactinantigens, it also provided better protection in the intracerebralchallenge test than a chemically detoxified component pertussis vaccine(ref. 30). Other suggested formulations include a formaldehyde-treatedK9G129 component (ref. 31) and a cellular vaccine derived from a strainof Bordetella pertussis producing the genetically inactivated K9G129pertussis toxin molecule (ref. 32). The formaldehyde treatment of theK9G129 molecule altered the immunogenicity of the molecule as loweramounts of specific antibodies were induced. The protective ability ofthe molecule was also decreased as it was less effective in theintracerebral challenge assay (ref. 32). However, the recombinantcellular vaccine derived from the K9G129 producing strain proved to beas effective as the whole cell pertussis vaccine (ref. 32).

Although the preceeding formulations demonstrate the advantages ofimproved safety and efficacy associated with the use of a geneticallydetoxified pertussis toxin molecule, they do not address the adverseeffects of DPT (diphtheria, pertussis and tetanus) vaccination notassociated with the pertussis molecule component (refs. 33, 46). All ofthe stated formulations involved the use of either 0.3 mg of aluminumphosphate (ref. 32) or 0.5 mg aluminum hydroxide (refs. 29, 30).Aluminum salts were introduced into the DT and DPT vaccine formulationsas an adjuvant that would potentiate strong antibody responses when thelevels of the toxoids or the numbers of Bordetella pertussis organismswere decreased to avoid adverse reactions (refs. 34, 35) and alum is nowroutinely used in these vaccines as an adjuvant. However, years of fieldexperience with these adsorbed pertussis vaccines and studies (refs. 36,37) have demonstrated that, although they contained less of theidentified reactogenic vaccine components, local reactions werenonetheless precipitated (refs. 38, 39, 40, 41, 42). Histopathologicalexamination of local abscesses produced following vaccination revealedaluminum hydroxide inclusions in giant cells (ref. 38). Investigationinto the frequency of such granulomas indicated that they wereassociated with the aluminum content in the vaccine as placebo immunizedgroups which received only the aluminum fraction of the vaccine,exhibited abscess formation at a similar reaction rate (ref. 43).Further evidence in support of the role of aluminum in these localreactions was derived from studies comparing aluminum adjuvant adsorbedand plain cholera and tetanus vaccines (refs. 44, 45). Deep innoculationof the vaccine into the muscle decreases the incidence of theseabscesses but although improved techniques can prevent the formation ofabscesses (ref. 39), the potentiation of IgE responses by aluminum saltsis not affected.

It would be advantageous to provide immunogenic compositions havingmodulated immune responses to the constituent antigens without thedisadvantages of local toxicity and contribution to hypersensitivity ofprior art extrinsic adjuvants.

SUMMARY OF INVENTION

The present invention relates to avoiding the problems associated withthe use of alum as an adjuvant in immunogenic compositions by employinga genetically-detoxified pertussis holotoxin, which itself may beimmunogenic, to effect modulation of an immune response to anon-Bordetella antigen.

While the elimination of alum from vaccine formulations could have beenan approach to address to the problems associated therewith, as notedabove, alum was included in vaccine formulations to provide an enhancedimmune response to the antigens in the formulation. Elimination of alum,therefore, would be expected to lead to a less effective formulation andwould be unlikely to have been proposed.

However, the genetically-detoxified pertussis holotoxin surprisinglyprovides a modulation of the immune response of a non-Bordetella antigenwhich enables vaccine formulations and other immunogenic compositions tobe provided which exhibit immune responses at least equivalent to thoseachieved by adjuvanting with alum.

Accordingly, in one aspect of the present invention, there is providedan immunogenic composition, which comprises a genetically-detoxifiedpertussis holotoxin, and at least one other, non-Bordetella, antigen,wherein said genetically-detoxified pertussis holotoxin is present in anamount sufficient to modulate an immune response to said other antigenin the absence of an extrinsic adjuvant.

The immune response which is modulated by the presence of thegenetically-detoxified pertussis holotoxin may be humoral and/or acellular immune response. In particular, the modulated immune responsemay be an enhanced IgG and/or cellular response to the other antigen.

The at least one other, non-Bordetella antigen present in theimmunogenic composition may provide a protective immune response to atleast one pathogen, which may be a bacterial, viral or parasiticpathogen. The antigen may be selected from a wide range of pathogens.Representative pathogens include Corynebacterium diphtheriae,Clostridium tetani, paramyxoviridae, haemophilus, influenza, hepatitis,meningococci, streptococci, schistosoma and trypanosome. The antigenalso may be selected from cancer-associated antigens, particularlymelanoma, bladder, lung, cervical and prostate cancer antigens.

The at least one other non-Bordetella antigen may comprise inactivatedtumor cells or membrane fractions thereof. Tumor cells may be removedfrom a cancerous host and then inactivated in any convenient manner, forexample, by irradiation or chemical inactivation. The inactivated cellsand/or membrane fraction thereof then are mixed with thegenetically-detoxified holotoxin to provide an immunogenic compositionaccording to the invention. Such composition then may be administered toa naive (i.e. non-cancer burdened) host to confer prophylacticprotection against tumor development. In addition, such composition maybe administered to a tumor-burdened host to promote an anti-tumor immuneresponse in the host.

The genetically-detoxified pertussis holotoxin may itself beimmunoprotective but the immunomodulating effect thereof may be obtainedin the absence of an immune response to the holotoxin. The provision ofgenetically-detoxified pertussis holotoxins is described in U.S. Pat.Nos. 5,085,862 and 5,221,618, assigned to the assignee hereof and thedisclosures of which are incorporated herein by reference.

The term “genetically-detoxified” as used herein has the same meaning asin the aforementioned U.S. Pat. Nos. 5,085,862 and 5,221,618, namely apertussis holotoxin mutant which exhibits a residual toxicity of about1% or less, preferably less than about 0.5% of that of the native toxin.The residual toxicity is determined by CHO cell clustering assay andADP-ribosyl-transferase activity.

Such genetically-detoxified pertussis holotoxin may be formed bymutagenesis of a nucleotide sequence coding for the holotoxin, asdescribed in the above-mentioned patents, so that at least one aminoacid is removed or replaced. Multiple amino acids also may be removed orreplaced.

The at least one amino acid which is removed or replaced may be presentin the S1 subunit, specifically ARG⁹, ARG¹³, TRP²⁶, ARG⁵⁸ and GLU¹²⁹.Where multiple amino acids are removed or replaced, it is preferred toremove or replace (S1)ARG⁹GLU¹²⁹. When such mutation is effected, it ispreferred to replace ARG⁹ by CYS⁹ and GLU¹²⁹ by GLY¹²⁹. (This specificmutant is sometimes depicted herein as K9G129.)

Below are Tables 1a and 2 containing details of several mutations ofpertussis holotoxin which may be used as the genetically-detoxifiedpertussis holotoxin in the immunogenic compositions provided herein.(The Tables appear at the end of the descriptive text). Table 1bcontains details of the in vivo characterization of the mutations ofTable 1a.

The immunogenic compositions of the invention may contain at least oneadditional Bordetella antigen, including agglutinogens, FHA andpertactin.

The immunogenic compositions provided herein may be formulated in thesubstantial absence of an extrinsic adjuvant as a vaccine for human oranimal administration. Such vaccine composition may exhibit a decreasedIgE response.

In one embodiment of the invention, the immunogenic compositions of theinvention may be formulated in the substantial absence of alum as amultivalent vaccine comprising the genetically-detoxified pertussisholotoxin in an immunoprotective form and amount along with diphtheriatoxoid and tetanus toxoid as the other antigens, thereby providing a DTPvaccine formulation from which alum or other extrinsic adjuvant isabsent. Such DTP vaccine formulations usually also contain otherBordetella antigens, including agglutinogens, FHA and pertactin.

In another aspect, the present invention provides a method of obtaininga modulated immune response to an antigen in a host, including a human,which comprises administering at least one non-Bordetella antigen to thehost, and coadministering to the host a genetically-detoxified pertussisholotoxin in an amount sufficient to modulate an immune response to theother antigen in the absence of an extrinsic adjuvant.

As noted above, the immune response may be a humoral and/or a cellularimmune response and the modulated immune response may be an enhanced IgGand/or cellular immune response. The administration of the pertussisholotoxin and other antigen may be effected by administering to the hosta composition as described above and provided according to theinvention.

In a particular embodiment of the present invention antigens andadjuvants are coadministered. In this application the term“coadministration” means simultaneous administrations or administrationswithin a short time period such as between several minutes or hours andup to 3 days. The coadministrations may be at the same or differentsites and by the same or different routes.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be further understood from the followingdescription with reference to the Figures in which:

FIG. 1 shows the potentiation of murine serum IgE antibody production byimmunogenic compositions of the present invention;

FIG. 2 shows the production of IgG antibodies by immunogeniccompositions of the present invention;

FIG. 3 shows the production of IgG antibodies by multivalent vaccines ofthe present invention; and

FIG. 4 shows the induction of cellular immune responses by multivalentvaccines of the present invention;

FIG. 5 shows the T_(h)1 and T_(h)2 immune response phenotypes, asdetermined by cytokine profile in mice immunized with ovalbuminadjuvanted with PPD and genetically detoxified pertussis toxin PT(K9G129);

FIG. 6 shows the T_(h)1 and T_(h)2 immune response phenotypes asdetermined by ovalbumin specific IgG2a and IgGE immunoglobulin profilesin mice immunized with PPD and genetically detoxified pertussis toxin,PT (K9GK9);

FIG. 7 shows the number of tumor-free mice in immunotherapy experimentsconducted herein. Six groups of mice with five mice per group wereimmunized with cell culture medium as a control and 10⁴ live melanomacells. The graph shows the number of mice that had no tumor thirty daysafter the challenge;

FIG. 8 shows the tumor volumes as at day 30 from two of the six groupsof mice plotted versus the number of days after challenge with 10⁵ livemelanoma cells. The open boxes with the dashed lines represent the fivemice that were immunized with 10⁴ irradiated cells alone (group 2) andthe closed circles connected by the solid lines represent the five miceimmunized with irradiated cells plus 1 μg of K9G129 (group 3); and

FIG. 9 shows the tumor volumes at day 22. Mice were immunized with cellculture medium as a control and 10⁴ irradiated melanoma cells alone ortogether with CFA or increasing concentrations of K9G129. The tumorvolumes of each of five mice in the six groups are shown 22 days afterchallenge with 10⁵ live melanoma cells. for the last three groups (micereceiving K9G129), the numbers by the arrows show the number of micethat were free tumors.

GENERAL DESCRIPTION OF INVENTION

Referring to FIG. 1, there is illustrated the potentiation of serum IgElevels in mice immunized with a chemically inactivated acellularalum-adjuvanted DTP vaccine and a non-alum adjuvanted rDTP acellularvaccine comprising the genetically-detoxified K9G129 PT analog. Theresults indicate that the serum IgE levels in mice immunized with therDTP acellular vaccine were significantly decreased (p<0.05) relative tothe DTP acellular vaccine containing the chemically toxoided pertussistoxin molecule.

Referring to FIGS. 2 and 3 and Table 3, there is illustrated acomparison of antigen specific antibody levels produced followingimmunization of mice with an alum adjuvanted DTP whole cell vaccine, analum-adjuvanted DTP acellular vaccine preparation and a DTP vaccinecontaining the genetically-detoxified PT analog K9G129 not adjuvantedwith alum. The results shown in Table 3 indicate that the anti-PT IgGresponse and the CHO neutralization titres produced by thealum-adjuvanted DTP acellular vaccine and the non-alum-adjuvantedrecombinant DTP acellular vaccine are equivalent. Thus, although thealum-free recombinant formulation demonstrated decreased IgEpotentiating activity, it nonetheless retained its effectiveness as apertussis vaccine as indicated by these anti-pertussis toxin IgG titres.Further evidence of the retention of PT-specific immunogenicity wasobtained from CHO cell neutralization assays. Significantly, higherlevels of anti-agglutinogen 2+3 and anti-69 kD (pertactin) IgGantibodies were detected in the serum samples from mice immunized withalum-free recombinant formulation (p<0.05). The anti-FHA toxoid IgGresponses were equivalent in sera obtained from mice immunized witheither of the vaccines.

FIG. 3 shows the anti-tetanus toxoid and anti-diphtheria toxoid IgGantibody levels in sera of mice immunized with either the whole cellpertussis vaccine, the defined component acellular DTP vaccinecontaining the glutaraldehyde-detoxified pertussis molecule or thealum-free recombinant acellular DTP vaccine. The diphtheria and tetanustoxoid components in the latter vaccine were also devoid of alum. Theresults indicate that the acellular formulations induce significantlyhigher anti-tetanus toxoid and particularly anti-diphtheria toxoid IgGantibodies as measured by this assay. Furthermore, the alum-freerecombinant formulation induced significantly higher anti-tetanus anddiphtheria toxoid IgG responses relative to the acellular component DTPvaccine.

The ability of the DTP vaccine formulations to induce antigen-specificcellular immune responses was evaluated in vitro and the results areshown in FIG. 4. Splenocytes derived from mice immunized with either thewhole cell, acellular or alum-free recombinant acellular DTP vaccineswere cultured in the presence of the specific vaccine antigens. Thewhole cell DTP vaccine induced a significant anti-diphtheria toxoidcellular response although not to the same degree as that generated bythe acellular component DTP vaccine. The acellular component vaccineinduced a relatively poor pertussis antigen specific proliferativeresponse with the exception of the anti-69 kD and anti-diphtheria toxoidresponses. Of significance, however, was the markedly increasedantigen-specific proliferative index induced by the alum-freerecombinant acellular formulation in response to all the antigenstested. The recombinant formulation clearly induced the highest levelsof antigen-specific proliferative responses of any of the vaccinestested.

In accordance with an embodiment of the invention there is provided (asan example of an immunogenic composition comprising agenetically-detoxified pertussis holotoxin and at least one othernon-Bordetella antigen wherein said genetically-detoxified pertussisholotoxin is present in an amount sufficient to modulated an immuneresponse to said other antigen in the absence of an extrinsic adjuvant)an alum-free acellular DPT vaccine containing the genetically-detoxifiedPT analog K9G129. Thus, although the alum-free formulation does notcontain an extrinsic (e.g. a mineral) adjuvant it does contain anadjuvant nonetheless as the K9G129 mutant acts not only as an antigenbut as an adjuvant (i.e. a proteinaceous adjuvant) as well. Thisproperty is apparent in the pertussis antigen specific responsesmeasured by enzyme immunoassay (FIG. 2). Significantly higheranti-agglutinogen 2+3 and anti-69 kD (pertactin) IgG responses wereevident in the serum samples derived from mice immunized with thealum-free recombinant acellular pertussis vaccine formulation while theFHA toxoid specific responses were equivalent. Therefore, the newformulation induced antibody responses specific for pertussis vaccineantigens at levels that were either comparable or greater than thelevels induced by the alum-adsorbed acellular pertussis vaccine.

The general intrinsic adjuvant activity of the K9G129 mutant for othervaccine antigens (such as those antigens present in human vaccines, suchas paediatric combination vaccines) was also evaluated. The tetanus anddiphtheria toxoid specific IgG responses in serum obtained from miceimmunized with either the alum adsorbed whole cell or acellularpertussis vaccines or the alum free recombinant vaccine were compared(FIG. 3). The tetanus and diphtheria specific IgG titres in the serum ofmice immunized with the whole cell vaccine were significantly lower thanthose observed in either of the acellular DTP immunized groups. Althoughthe alum-adsorbed DTP vaccine induced significantly higher toxoidspecific responses relative to the whole cell vaccine immunized group,of all the vaccine formulations tested, the alum-free formulationinduced the highest titres of toxoid specific IgG. Therefore, theadjuvant activity of the K9G129 mutant is not restricted to onlyBordetella antigens. The invention extends to a multivalent vaccinecontaining protective antigens for a plurality of pathogens.

Vaccine Preparation and Use

As indicated above, the present invention in one embodiment providesimmunogenic compositions, suitable to be used as, for example, vaccines.The immunogenic composition elicits an immune response by the host towhich it is administered including the production of antibodies by thehost. The immunogenic compositions include at least one non-Bordetellaantigen in one embodiment. This antigen may be an inactivated pathogenor an antigenic fraction of a pathogen. The pathogen may be, forexample, a virus, a bacterium or a parasite. The pathogen may beinactivated by a chemical agent, such as formaldehyde, glutaraldehyde,β-propiolactone, ethyleneimine and derivatives, or other compounds. Thepathogen may also be inactivated by a physical agent, such as UVradiation, gamma radiation, “heat shock” and X-ray radiation.Representive pathogens from which the antigen may be derived includeCorynebacterium diphtheriae, Clostridium tetani, paramyxoviridae,haemophilus, influenza, hepatitis, meningococci, streptococci,schistosoma and trypanosome.

An antigenic fraction of a pathogen can be produced by means of chemicalor physical decomposition methods, followed, if desired, by separationof a fraction by means of chromatography, centrifugation and similartechniques. In general, low molecular components are then obtainedwhich, although purified, may have low immunogenicity, alternativeantigens include cancer-specific antigens including melanoma, lung,cervical, prostate and bladder cancer antigens. Alternatively, antigensor haptens can be prepared by means of organic synthetic methods, or, inthe case of, for example, polypeptides and proteins, by means ofrecombinant DNA methods.

The immunogenic compositions may be prepared as injectables, as liquidsolutions or emulsions. The antigens and immunogenic compositions may bemixed with physiologically acceptable carriers which are compatibletherewith. These may include water, saline, dextrose, glycerol, ethanoland combinations thereof. The vaccine may further contain auxiliarysubstances, such as wetting or emulsifying agents or pH bufferingagents, to further enhance their effectiveness. Vaccines may beadministered by injection subcutaneously or intramuscularly.

Alternatively, the immunogenic compositions provided by the presentinvention, may be delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal, anal, vaginal or oral(intragastric) routes. Alternatively, other modes of administrationincluding suppositories may be desirable. For suppositories, binders andcarriers may include, for example, polyalkylene glycols andtriglycerides. Oral formulations may include normally employedincipients, such as pharmaceutical grades of saccharine, cellulose andmagnesium carbonate.

These compositions may take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and contain 1to 95% of the immunogenic compositions of the present invention.

The immunogenic compositions are administered in a manner compatiblewith the dosage formulation, and in such amount as to be therapeuticallyeffective, protective and immunogenic. The quantity to be administereddepends on the subject to the immunized, including, for example, thecapacity of the subject's immune system to synthesize antibodies, and ifneeded, to produce a cell-mediated immune response. Precise amounts ofantigen and immunogenic composition to be administered depend on thejudgement of the practitioner. However, suitable dosage ranges arereadily determinable by those skilled in the art and may be of the orderof micrograms to milligrams. Suitable regimes for initial administrationand booster doses are also variable, but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and will varyaccording to the size of the host.

The concentration of antigens in an immunogenic composition according tothe invention is in general 1 to 95%. A vaccine which contains antigenicmaterial of only one pathogen is a monovalent vaccine. Vaccines whichcontain antigenic material of several pathogens are combined vaccinesand also belong to the present invention. Such combined or multivalentvaccines contain, for example, material from various pathogens or fromvarious strains of the same pathogen, or from combinations of variouspathogens.

Immunoassays

In one embodiment, the immunogenic composition of the present inventionare useful for the generation antigen-specific antibodies that arethemselves useful in the specific identification of that antigen in animmunoassay. Such immunoassays include enzyme-linked immunosorbentassays (ELISA), RIAs and other non-enzyme linked antibody binding assaysor procedures known in the art. In ELISA assays, the antigen-specificantibodies are immobilized onto a selected surface; for example, thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed antibodies, a nonspecific protein, such as asolution of bovine serum albumin (BSA) or casein, that is known to beantigenically neutral with regard to the test sample may be bound to theselected surface. This allows for blocking of nonspecific adsorptionsites on the immobilizing surface and thus reduces the background causedby nonspecific bindings of antigens onto the surface. The immobilizingsurface is then contacted with a sample, such as clinical or biologicalmaterials, to be tested in a manner conducive to immune complex(antigen/antibody) formation. This may include diluting the sample withdiluents, such as BSA, bovine gamma globulin (BGG) and/or phosphatebuffered saline (PBS)/Tween. The sample is then allowed to incubate forfrom about 2 to 4 hours, at temperatures such as of the order of about25° to 37° C. Following incubation, the sample-contacted surface iswashed to remove non-immunocomplexed material. The washing procedure mayinclude washing with a solution such as PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the antigen inthe test sample and the bound antigen-specific antibodies, andsubsequent washing, the occurrence, and even amount, of immunocomplexformation may be determined by subjecting the immunocomplex to a secondantibody having specificity for the antigen. To provide detecting means,the second antibody may have an associated activity, such as anenzymatic activity, that will generate, for example, a colourdevelopment upon incubating with an appropriate chromogenic substrate.Quantification may then achieved by measuring the degree of colourgeneration using, for example, a visible spectra spectrophotometer.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Example 1

This Example describes the formulation of vaccines.

The DPT whole-cell vaccine and an experimental component acellularvaccine were produced by Connaught Laboratories Ltd. The componentacellular pertussis vaccine was alum adsorbed (1.5 mg/dose) andconsisted of 10 μg protein nitrogen of glutaraldehyde toxoided pertussistoxin, 5 μg protein nitrogen each of FHA and agglutinogens 2 and 3 and 3μg of pertactin along with 5 Lf of tetanus toxoid and 25 Lf ofdiphtheria toxoid per dose. The recombinant component vaccine alsocontained 5 μg protein nitrogen each of FHA and agglutinogens 2 and 3and 3 μg protein nitrogen of pertactin in addition to 5 Lf of tetanustoxoid and 25 Lf of diphtheria toxoid per dose. However, it varied fromthe other acellular vaccine in that it contained 20 μg protein nitrogenof the recombinant PT mutant holotoxin, K9G129, and was not alumadsorbed. The K9G129 pertussis toxin molecule as well as the purifiedFHA, agg 2+3 and pertactin components were obtained individually fromConnaught Laboratories Ltd.

Example 2

This Example describes immunization of animals.

Female BALB/c mice weighing 15 to 18 grams were obtained from CharlesRiver Canada (St. Constant, Quebec). The mice were housed inmicroisolators and used in accordance with the guidelines set by theCanadian Council on Animal Care (CCAC). The animals were specificpathogen free and the housing rooms were monitored for Murine HepatitisVirus outbreaks through the use of sentinel mice. Water was provided adlibitum and the diet was ovalbumin-free. The mice were immunized on Day0 with the vaccine formulations in groups of six. A booster dose ofvaccine was administered on Day 21. On Day 28 the animals were bled viajugular vein laceration and splenectomized. The serum samples werestored at −20° C. until assayed.

Example 3

This Example describes antigen specific immunoassays.

The vaccine antigen specific IgG responses were determined by indirectEIA. The antigens of interest were pertussis toxin, pertactin,filamentous hemagglutinin, agglutinogens, as well as diphtheria andtetanus toxoids. High binding capacity microplates (Nunc) were coatedwith 4 μg/mL of each of the above antigens in a volume of 50 uls/well of50 mM carbonate buffer pH 9.6. After an overnight incubation, the plateswere washed and successively blocked with a 0.1% solution of bovineserum albumin (Sigma) for one hour at room temperature. The excess blockwas removed and the microplates were washed. The murine serum sampleswere then serially diluted in PBS-Tween 20 (0.05%) and plated out at avolume of 100 uls. The samples were incubated overnight at 4° C. Theantigen specific fraction of IgG antibodies was detected by a peroxidaseconjugated sheep anti-mouse IgG conjugate (Jackson Laboratories). Theplates were developed with the TMB substrate as above and were read atdual wavelengths of 450 nm and 540 nm in the Multiskan MCC 340 MkIImicroplate reader. Reactive titres were defined as the dilution at whichthe absorbance of the test sample was equivalent to the mean plus threestandard deviations of the negative control absorbance values. Thegeometric means and 95% confidence intervals were calculated and thegroups were compared using the Student's t-test.

Example 4

This Example describes the determination of ovalbumin specific IgGsubclass profiles.

Example 5

This Example describes total IgE immunoassays.

The serum total IgE levels were assessed by indirect EIA. Nuncimmunoplates (Gibco/BRL) were coated at room temperature overnight witha sheep anti-mouse IgE polyclonal antiserum (Serotec) diluted in 50 mMcarbonate buffer pH 9.6. The next day the plates were washed in PBScontaining 0.05% Tween 20 (J. T. Baker) and then blocked with 0.1%casein amino acids (Difco) for one hour at room temperture. After theexcess blocking solution was washed off the plates, the murine serumsamples were serially diluted three-fold in the assay diluent and platesout onto the microplate at 100 uls per well. The samples were incubatedovernight at 4° C. To detect the bound IgE antibodies, a biotinylatedrat anti-mouse Ige monoclonal antibody (Serotec) was added to each wellat a concentration of 2 ug/mL and incubated for one hour at roomtemperature. After washing, peroxidase conjugated streptavidin(Dimension Laboratories) was added to each well. The amount of IgE boundto the wells was assessed by adding the enzyme substrate, 10%tetramethylbenzidine (TMB) (ADI Diagnostics) in 0.005% hydrogen peroxidewater (Fisher Scientific). The reaction was stopped after ten minuteswith 1M sulfuric acid (Fisher Scientific). The absorbance of the wellswas measured at 450 nm with a background correction at 540 nm on aMultiskan MCC 340 MkII microplate reader (Flow Laboratories). The serumIgE levels were quantitated by calibrating the sample absorbancesagainst a standard curve generated by a serially diluted IgE murinemyeloma protein run on each plate. The geometric means and 95%confidence intervals were calculated for each treatment group and thegroups were compared using the Student's t-test and p<0.05.

Example 6

This Example describes the determination of ovalbumin specific IgGFimmunoglobulins.

The levels of IL-4 and IFN-γ were determined in a sandwich EIA. Briefly,96 well Nunc Maxisorp microplates (Gibco/BRL) were coated overnight atroom temperature with cytokine monospecific rat monoclonal antibodies.These antibodies were obtained from Pharmingen and derived from thefollowing respective clones: IL-4, clone 11B11; IL-5; IFN-γ, cloneR4-6A2. The monoclonal antibodies were diluted to a concentration of 2μg/mL in 50 mM carbonate buffer pH 9.6. The following day, the plateswere washed in PBS-Tween 20 0.05% (PBS-T) and nonspecific binding siteswere blocked by the addition of a 1% bovine serum albumin (Sigma)solution diluted in PBS-T. Following incubation for one hour at roomtemperature, the excess block was washed from the plates and undilutedculture supernatants were added to the wells in duplicate. Theappropriate recombinant standards for each cytokine (recombinant IL-4obtained from Pharmingen, recombinant IFN-γ purchased from Genzyme) werediluted to the appropriate concentrations (initial concentration of 100ng/mL or 1000 ng/mL for IL-10 EIA) and serially diluted three-fold inRPM 1640 (Sigma) containing 10% fetal bovine serum (FBS). The standardswere plated out at 100 μls/well and the microplates were incubatedovernight at 4° C. After a vigorous wash in PBS-T, the bound cytokineswere detected using a biotinylated monoclonal antibody specific to eachcytokine and diluted to a concentration of 2 μg/mL in PBS-T. Theantibodies were obtained from Pharmingen and were derived from thefollowing clones: IL-4 clone BVD6-24G2; IFN-γ, clone XMG1.2. After a onehour incubation step at room temperature, a peroxidase conjugatedstreptavidin preparation (Vector Laboratories) diluted to aconcentration of 500 ng/mL was added. A final wash was performedfollowing a one hour incubation at room temperature of the streptavidinpreparation. The plates were developed by the addition of the substrate,10% TMB in 0.05% hydrogen peroxide (Fisher Scientific) water. Thereactions were allowed to proceed until suitable colour intensity wasreached and were stopped by the addition of 100 μls/well of a 1Msolution of sulfuric acid (Fisher Scientific). The absorbances of thereaction wells were read at dual wavelengths (450 nm and 540 nm) on aMultiskan MCC 340 MkII (Flow Laboratories) microplate reader.

The cytokine concentrations in the supernatants were quantitated bycalibrating the sample absorbances against the absorbances of thestandards of known concentrations using the logistic curvefit algorithmto fit the curve with a minimum correlation coefficient of 99.9%. TheELISA+ software package (Meddata) was used to quantitate the amounts ofcytokines present in the supernatants based on the standard curvesgenerated on each plate.

Ovalbumin-specific IgE titres in the sera of immunized mice weredetermined by use of an indirect antigen capture EIA. Briefly, NuncMaxisorp microplates (Gibco/BRL) were coated with a rabbitanti-ovalbumin IgG fraction (Cappel Laboratories). The plates wereincubated overnight at room temperature. The next day, after washing inPBS-T, the plates were blocked with a solution of 0.1% skimmed milkpowder diluted in PBS-T for one hour at room temperature. Next, asolution of ovalbumin diluted to 10 μg/mL in 50 mM carbonate buffer pH9.6 was added to each well in 100 μl volumes. Following a one hourincubation at room temperature, the ovalbumin solution was washed offthe plates. The murine serum samples were then serially dilutedthree-fold in PBS-T at an initial dilution of 1:40 and a final dilutionof 1:87480. 100 ml samples were added per well and incubated overnightat 4° C. After washing the next day, the ovalbumin specific IgEantibodies bound to the plates were detected using a biotinylated ratanti-mouse IgE monoclonal antibody (Clone LO-ME-2, Serotec) diluted to 2μg/mL in PBS-T. Following a further one hour incubation at roomtemperature, this antibody was washed off and peroxidase conjugatedstreptavidin (Vector Laboratories) was added to each well at aconcentration of 500 ng/mL. The amount of ovalbumin-specific IgE in themurine serum samples was detected by adding the peroxidase substrate,10% tetramethylbenzidine (TMB) (ADI Diagnostics) in 0.005% hydrogenperoxide. The color in the wells was allowed to develop for fifteenminutes and the reactions were stopped by the addition of 100 μls of 1Msulfuric acid (Fisher Scientific). The absorbance of the wells wasmeasured in a microplate reader (Multiskan MCC 340 Mk11, FlowLaboratories) at 450 nm with a reading at 540 nm for backgroundcorrection. Reactive titres were defined as the last dilution at whichthe absorbance value of the test sample was equivalent to the mean ofthe absorbance values derived from a negative serum control plus threestandard deviations. The geometric means were calculated on logtransformed data and expressed with 95% confidence intervals.

Example 7

This Example describes the determination of murine cytokine profiles.

The ovalbumin-specific IgG, IgG1 and IgG2a titres in murine serumsamples were measured by indirect EIA. In the IgG2a assay, Nuno Maxisorp96-well microplates (Gibco/BRL) were coated with a rabbit anti-ovalbuminpolyclonal antibody IgG fraction (Cappell Laboratores) diluted in 50 mMcarbonate buffer pH 9.6 and incubated overnight at room temperature.Ovalbumin-specific IgG and IgG1 responses were measured on microplatescoated directly with ovalbumin (Sigma) diluted to a concentration of 10μg/mL in 50 mM carbonate buffer. The following day, the microplates werewashed in PBS-T and blocked for one hour at room temperature with asolution of 0.1% skimmed milk powder diluted in PBS-T. After a furtherwashing step, a 10 μg/mL solution of ovalbumin (Sigma) diluted in 50 mMcarbonate buffer pH 9.6 was added to the IgG2a specific assay. Thisantigen coat was incubated for one hour at room temperature and wasfollowed by a washing step.

The next step in the assay required the addition of the murine serumsamples. In the IgG2a assay, the serum samples were serially dilutedthree-fold beginning at an initial dilution of 1:40 and ending at adilution of 1:87480. The IgG and IgG1 assays were carried out with serumsamples diluted three-fold starting at an initial dilution of 1:360 andending at a final dilution of 1:787320. The serum samples were dilutedin PBS-T and added to the wells of the microplates in 100 μl volumes.The plates were incubated overnight at 4° C. The next day, the plateswere washed and the ovalbumin-specific IgG subclasses of antibodies weredetected with biotinylated rat anti-mouse IgG conjugates specific foreach IgG antibody subclass (IgG2a conjugate, derived from clone R19-15and obtained from Pharmingen, IgG1 conjugate, derived from cloneLO-MGI-2 and obtained from Serotec) while the IgG responses weredetected with a 1:50,000 dilution of a peroxidase-labelled sheepanti-mouse IgG (Fcγ specific, Jackson Laboratories). The conjugatedmonoclonal antibodies were diluted to a concentration of 2 μg/mL andincubated for one hour at room temperature. After washing, peroxidaseconjugated streptavidin was added to each well of the plates containinga biotinylated conjugate at a concentration of 500 ng/mL. The plateswere incubated for one hour at room temperature. The boundantigen-specific IgG and IgG subclass antibodies were detected by theaddition of the peroxidase substrate, 10% TMB (ADI Diagnostics) dilutedin 0.005% hydrogen-peroxide (Fisher Scientific). The reactions wereallowed to proceed for a period of ten minutes at which point they wereterminated by the addition of 1M sulfuric acid (Fisher Scientific) toeach well. The microplates were read on a microplate reader (MultiskanMCC 340 MkII, Flow Laboratories) at dual wavelengths of 450 nm and 540nm. Reactive titres were defined as the last dilution at which theabsorbance of the test sample was equivalent to the mean plus threestandard deviations of the negative control absorbance values. Thegeometric means were calculated on log transformed data and expressedwith 95% confidence intervals.

Example 8

This Example describes antigen-specific cellular immune responses.

Murine splenocytes were obtained from the vaccine immunized BALB/c miceon Day 28. The spleens were dissociated into a single cell suspensionand washed three times in RPMI 1640 media (Sigma). A cell count wasperformed using the trypan blue exclusion method and the cells wereadjusted to a concentration of 2×10⁶ cells/mL. The antigens (pertussistoxoid, pertactin, FHA, agglutinogens, and non alum-adsorbed diphtheriaand tetanus toxoids were diluted to a concentration of 5 μg/mL in RPMI1640 media containing 10% fetal bovine serum. The antigens were thenserially diluted two fold to a concentration of 78 ng/mL. The cells werethen added to each well at a final concentration of 1×10⁵ cells/well.The cultures were left to incubate at 37° C. in a 5% CO₂ incubator for72 hours. At the end of this period, the cells were pulsed with 0.5μCi/well of tritiated thymidine (Amersham) diluted in sterile PBS(Sigma). After a further 18 hour incubation, the cells were harvestedonto glass fibre filter paper using a 96 well harvester (CanberraPackard) and the radioactive counts were read on a Matrix 96 betacounter (Canberra Packard. The results were expressed as stimulationindices which were calculated by dividing the means of the test countsby the means of the background counts on the plate. Each sample wasassayed in triplicate.

Example 9

This Example describes the ability of antibodies to neutralize pertussistoxin in the CHO cell neutralization assay.

The ability of the antibodies induced by the pertussis vaccines toneutralize pertussis toxin was assessed in the CHO cell assay asdescribed by Granstrom et al. (ref. 47). The last dilution of antibodyat which no significant morphological effects could be seen was definedas the neutralizing titer. The results were expressed as reciprocalneutralizing titers.

Example 10

This example illustrates the use of genetically-detoxified pertussisholotoxin to confer prophylactic protection against tumor development.

Thus, the B16 mouse melanoma model (Ref. 54) was used to assess theeffectiveness of K9G129 as an adjuvant in cancer immunotherapy. WhenC57Bl/6 mice were injected subcutaneously with live syngeneic B16-F1strain of B16 melanoma cells, tumors appeared after about ten days andprogressively grew in an exponential manner. Tumor appearance wasdirectly proportional to the dose of cells injected, eg. tumors formedearlier when mice were injected with 10⁶ cells than with 10⁴ cells.Tumors could be delayed by immunizing the mice with B16 melanoma cellsthat had first been irradiated with 10,000 rads. This delay was alsodose dependent. Immunizing with 10⁶ irradiated cells caused a greaterdelay in tumor appearance than immunizing with 10⁵ irradiated cells,when the mice were subsequently challenged with 10⁵ live cells.Immunizing with 10⁴ irradiated cells caused no significant delay intumor growth.

The effectiveness of K9G129 as an adjuvant was tested by measuring itsability to delay tumor growth when combined with 10⁴ irradiated cells inan immunization experiment. Six groups of mice with five mice per groupwere immunized with:

1) cell culture medium (control)

2) 10⁴ irradiated cells

3) 10⁴ irradiated cells+1 μg K9G129

4) 10⁴ irradiated cells+5 μg K9G129

5) 10⁴ irradiated cells+10 μg K9G129

6) 10⁴ irradiated cells+CFA (complete Freunds adjuvant)

The mice were boosted in the same manner two weeks later and then twoweeks after the boost they were challenged with 10⁵ live B16 melanomacells. The appearance of tumors was monitored and the size of growingtumors was measured with calipers, noting both the length and width. Thevolume of the tumors was calculated by applying these measurements tothe formula for an ellipsoid.

K9G129 was effective in a dose dependent manner in delaying the onset oftumor growth. Thirty days after the challenge with 10⁵ live melanomacells, there were no mice without tumors in the groups that received noimmunization (group 1), irradiated cells alone (group 2), or irradiatedcells with CFA (group 6). There were also no tumor-free mice in thegroup that had received irradiated cells with 1 μg of K9G129 (group 3).However there were two and four mice respectively that had no tumor fromthe groups that had received irradiated cells with 5 μg and 10 μg ofK9G129 (groups 4 and 5) (FIG. 7). These results show a large delay intumor appearance mediated by the two higher concentrations of K9G129.

Even the lowest concentration of K9G129 used caused a delay in tumorappearance. Four of five mice in group 3 (irradiated cells+1 μg K9G129)had tumors appear after tumors had begun to grow in the mice that hadbeen immunized with irradiated cells alone (group 2) (FIG. 8). Theeffectiveness of K9G129 in delaying tumor growth is further demonstratedby comparing the tumor volumes of individual mice in the various groups,22 days after the challenge with live melanoma cells. Tumors arenon-existent or their sizes are generally lower in mice that wereimmunized with irradiated cells and K9G129 than in mice immunized withirradiated cells alone or in conjunction with CFA (FIG. 9).

These results indicate that K9G129 can act as an adjuvant in cancerimmunotherapy to increase the immune response towards tumor cells.

Example 11

This Example describes the generation of a Th1 response to an immunogenadjuvanted with the S1(K9G129) Pertussis Toxin analogue.

One, of the key factors involved in the potentiation of differentimmunoglobulin subclasses, including IgE, is the presence of solublemediators known as cytokines. The control of IgE production is regulatedby a variety of cytokines which not only possess direct effectorfunctions such as the induction of immunoglobulin isotype switching, butalso act to cross-regulate the production of other cytokines. In themouse, IL-4 acts not only to induce IgE and IgG1 isotype switching, butalso acts to inhibit the secretion of IgM, IgG3, IgG2b and IgG2a (Ref.48). On the other hand, IFN-γ acts to stimulate the production of IgG2aand IgG3 while inhibiting IgG1, IgG2b and IgE synthesis (Ref. 48).

In an effort to organize and rationalize the multiple andcross-regulatory effects of cytokines, a system to describe the variouspatterns of cytokine secretion has been described (Ref. 49). Mosmann andCoffman (Ref. 49) defined two distinct subsets of murine CD4⁺ T cellsbased on their differential patterns of cytokine secretion. Using longterm T cell clones, they were able to show that one group of cells,defined as Th2, secreted IL-4, IL-5 and IL-10 while another group ofcells, defined as Th1, secreted IL-2, IFN-γ and TNF-β. These twodistinct cytokine profiles were also correlated with immunoglobulinproduction in that Th1 clones provided help for B lymphocytes to produceIgG2a while Th2 clones promoted the secretion of IgG1 and IgE by B cells(48,50,51). Later work by Romagnani and coworkers demonstrated theexistence of these T cell subsets in humans as well (Ref. 52).

Although the initial differentiation of Th1/Th2 cytokine profiles wasdefined on the basis of in vitro cytokine patterns of individual T cellclones, the definitions have been extended to describe the cytokinephenotypes resulting from immunization or infection (Ref. 48,53). Thesephenotypes are not as starkly polar as those observed in the originalclonal analysis and are defined by a variety of different cytokines.Thus, a Th1 phenotypic response is characterized by a significantincrease in Th1-type cytokines (higher ratios of IFN-γ:IL-4) relative toTh2 immune response phenotypes. This classification also extends to theantigen-specific immunoglobin subclass profiles where Th1 phenotypespresent as higher IgG2a:IgE ratios relative to Th2 type, responses.

FIG. 5 shows the cytokine profile in mice immunized with ovalbumin andadjuvanted with PPD and PT(K9G129). PPD is an adjuvant that produces aTh1 immune response.

Splenocytes were obtained from mice immunized with ovalbumin along witheither S1(K9G129) rPT or PPD as adjuvants. The spleen cells of four micein each treatment group were pooled and then restimulated in vitro withovalbumin alone (no adjuvant). The supernatants were then harvested fromthese cultures and the levels of IFN-γ and IL-4 were determined by EIA.Similar IFN-γ:IL-4 ratios were obtained from cultures derived from miceimmunized with either S1(K9G129) or PPD as an adjuvant. As describedabove, a higher ratio of IFN-γ:IL4 cytokines is characteristic of a Th1immune response.

FIG. 6 shows the ovalbumin-specific IgG2a and IgE responses of BALB/cmice immunized with ovalbumin and either the S1(K9G129) PT analogue orPPD as adjuvants. The bar graph indicates that immunization with theS1(K9G129) PT analogue resulted in ratios of ovalbumin-specificIgG2a:IgE ratios similar to those obtained following immunization withPPD. As described above, a high IgG2a:IgE ratio is characteristic of aTh1 immune response. The results in FIGS. 5 and 6 thus indicate thatadjuvanting with PT(K9G129) produces a Th1 immune response in mice.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides novelimmunogenic compositions and methods of immunization in which agenetically-detoxified pertussis holotoxin, which may also beimmunogenic, is employed as a proteinaceous adjuvant in place ofconventional extrinsic adjuvants, particularly alum, to achieve amodulated immune response to a non-Bordetella antigen without theadverse side effects of alum. Modifications are possible within thescope of the invention.

TABLE 1a Mutations introduced into Pertussis Toxin Mu- tation NumberMutation 1. ARG⁹ −> ▴9 2. ″ −> GLU⁹ 3. ″ −> LYS⁹ 4. ″ −> HIS⁹ 5. ARG¹³−> ▴13 6. ″ −> GLU¹³ 7. ARG⁹–ARG¹³ −> ▴9–13 8. ARG⁹ ARG¹³ −> GLU⁹ GLU¹³9. ARG⁵⁸ −> GLU⁵⁸ 10. ARG⁵⁷ ARG⁵⁸ −> ▴57▴58 11. TRP²⁶ −> ALA²⁶ 12. ″ −>CYS²⁶ 13. CYS⁴¹ −> ALA⁴¹ 14. ″ −> SER⁴¹ 15. CYS²⁰¹ −> ALA²⁰¹ 16. GLU¹²⁹−> ▴129 17. ″ −> GLY¹²⁹ 18. ″ −> GLN¹²⁹ 19. ″ −> ASP¹²⁹ 20. ″ −> ASN¹²⁹21. ″ −> LYS¹²⁹ 22. ″ −> ARG¹²⁹ 23. ″ −> HIS¹²⁹ 24. ″ −> PRO¹²⁹ 25. ″ −>CYS¹²⁹ 26. ″ −> GLY¹²⁹ II 27. ″ −> GLN¹²⁹ II 28. TYR¹³⁰ −> ▴130 29. ″ −>PHE¹³⁰ 30. GLU¹²⁹ TYR¹³⁰ −> GLY¹²⁹ ALA¹³⁰ 31. GLU¹²⁹ TYR¹³⁰ −> GLN¹²⁹ALA¹³⁰ 32. GLU¹²⁹ TYR¹³⁰ −> GLY¹²⁹ PHE¹³⁰ 33. S3)LYS¹⁰ −> GLN¹⁰ 34.(S3)TYR⁹² LYS⁹³ −> ASN⁹² ARG⁹³ 35. (S3)LYS¹⁰⁵ −> ASN⁰⁵ 36. CYS⁴¹ CYS²⁰1−> ALA⁴¹ ALA²⁰¹ 37. CYS⁴¹ GLU¹²⁹ −> ALA⁴¹ GLY¹²⁹ 38. ARG⁹ GLU¹²⁹ −> GLU⁹GLY¹²⁹ II 39. ARG⁹ GLU¹²⁹ −> GLU⁹ GLN¹²⁹ II 40. ARG⁹ GLU¹²⁹ −> GLU⁹^(ARG) ¹²⁹ 41. ARG⁹ GLU¹²⁹ TYR¹³⁰ −> GLU⁹ GLY¹²⁹ ALA¹³⁰ 42. ARG¹³ GLU¹²⁹−> GLU¹³ GLY¹²⁹ II 43. ARG¹³ GLU¹²⁹ −> GLU¹³ GLN¹²⁹ II 44. ARG¹³ GLU¹²⁹TYR¹³⁰ −> GLU¹³ GLY¹²⁹ ALA¹³⁰ 45. ARG⁹ GLU¹²⁹ −> ▴⁹ GLN¹²⁹ S-2730-1-146. ARG⁹ GLU¹²⁹ TYR¹³⁰ −> ▴⁹ GLY¹²⁹ ALA¹³⁰ 47. ARG¹³ GLU¹²⁹ −> ▴¹³GLN¹²⁹ 48. ARG¹³ GLU¹²⁹ TYR¹³⁰ −> ▴¹³ GLY¹²⁹ ALA¹³⁰ 49. GLU¹²⁹ −> GLY¹²⁹(S3)TYR⁹² LYS⁹³ (S3)ASN⁹² ARG⁹³ 50. Wild Type 51. Arg13 −> Lys13 52.Arg58 −> His58 53. Arg58 −> Lys58 54. His35 −> Ala35 55. Glu129 −>Ser129 56. Tyr130 −> Ser130 57. Arg58Glu129 −> GLu58Gly129 58.Arg9Glu129 −> Lys9Gly129 59. Arg9Arg58Glu129 −> Lys9Glu58Gly129 60. (S3)Ile91Tyr92Lys93 −> Delete 61. (S2) Thr91Arg92Asn93 −> Delete 62. (S1)Glu129/ −> (S1) Gly129/ (S3) Ile91Tyr92Lys93 S3(91–93) delete 63. (S1)Arg58Glu129/ −> (S1) Glu58Gly129/ S3(91–93) S3(91–93) delete 64. (S1)Arg9Glu129/ −> (S1) Lys9Gly129/ S3(91–93) S3(91–93) delete 65. (S1)Arg9Arg58Glu129/ −> (S1) S3(91–93) Lys9Glu58Gly129 S3(91–93) delete 66.(S2) Thr91Arg92Asn93/ −> S2(91–93) delete S3(91–93) S3(91–93) delete 67.(S3) TYR⁸² −> ALA⁸² 68. (S3) ILE⁹¹ −> ▴⁹¹▴⁹²▴⁹³ 69. (S3) TYR¹⁰²TYR¹⁰³ −>A¹⁰²A¹⁰³ 70. (S3) LYS¹⁰⁵ −> ▴¹⁰⁵ 71. (S3) LYS¹⁰⁵ −> ALA¹⁰⁵ 72. (S3)LYS¹⁶⁹ −> ALA¹⁶⁹ 73. (S3) TYR⁸²LYS¹⁶⁹ −> ALA⁸²ALA¹⁶⁹ 74. (S4) TYR⁴ −>ALA⁴ 75. (S4) TYR²¹ −> ALA²¹ 76. (S4) LYS⁵⁴LYS⁵⁷ −> ALA⁵⁴ALA⁵⁷ 77. (S3)TYR⁸²(S4)LYS4⁵⁴LYS⁵⁷ −> (S3) ALA⁵²/ (S4)ALA⁵⁴ALA⁵⁷ 78. (S1)GLU¹²⁹/(S3)TYR⁸² −> (S1) GLY¹²⁹/ S3(▴82) 79. (S1)GLU¹²⁹/S3(ILE⁹¹TYR⁹²LYS⁹³) −> (S1) GLY¹²⁹/ (S3)▴⁹¹▴⁹²▴⁹³ Notes: Aminoacid numbering corresponds to positions in the native subunits. Allmutations are in subunit S1 unless specified as being in S2, S3 or S4.II denotes use of an alternative codon. ▴ denotes deleted residue(s).Wild type refers to PT expressed from the unmutated TOX operon in B.parapertussis.

TABLE 1b In vitro characterization of pertussis toxin analogues obtainedfrom recombinant B. parapertussis. Mutation Residual ADPR NumberToxicity (%) Activity (%) S1 Epitope 1. 0.2 ND − 2. 0.1 0.2 +/− 3. 0.1ND ++++ 4. 0.2 0.1 +++ 5. 0.3 ND − 6. 5.0 ND ++++ 7. 0.4 0.1 − 8. 0.10.9 − 9. 0.7 0.6 +++ 10. 0.4 ND − 11. 0.5 ND + 12. 6.0 ND ND 13. 0.3 0.4− 14. 1.4 ND ND 15. 0.2 0.1 − 16. 0.1 ND ++ 17. 0.1 0.3 ++++ 18. 0.020.1 +/− 19. 0.7 2.5 ++ 20. 0.1 0.3 ++ 21. 0.3 0.2 − 22. 0.1 ND − 23. 0.2ND − 24. 0.2 ND + 25. 0.4 ND − 26. 0.1 0.3 ++++ 27. 0.02 0.1 +/− 28. 0.20.1 − 29. 12.0 ND ++++ 30. 0.2 0.6 − 31. 0.4 ND − 32. 1.0 ND ++++ 33.100 ND ++++++ 34. 50 100 ++++ 35. 20 ND ++++ 36. 0.2 0.1 − 37. 0.1 0.1 −38. 0.1 0.1 − 39. 0.1 ND − 40. 0.1 ND − 41. 0.2 ND − 42. 0.5 ND − 43.3.0 ND − 44. 0.3 ND − 45. 0.4 ND − 46. 0.2 0.1 − 47. 0.5 ND − 48. 0.40.3 − 49. 0.2 0.1 ++++ 50. 100 100 ++++ 51. 14.0 +++++ 52. 35.0 +++++53. 13.0 +++++ 54. 0.2 ++ 55. 0.6 +++++ 56. 29.0 ++++ 57. 0.1 ++ 58.<0.001 <0.001 +++ 59. 0.1 + 60. 12.0 +++++ 61. 100.0 +++++ 62. 0.03 0.2+++ 63. 0.1 + 64. 0.1 +++ 65. 0.1 + 66. 10.0 + 67. 7.2 96 ND 68. 4.6 108ND 69. 9.6 98 ND 70. 8.1 57 ND 71. 94 71 ND 72. 102 71 ND 73. 5.2 92 ND74. 46 125 ND 75. 84 91 ND 76. 9.6 55 ND 77. 1.5 97 ND 78. 0.04 0.10 ND79. 0.04 0.16 ND Notes: Residual toxicity is the ratio of the apparentPT concentration determined by the CHO cell clustering assay to theactual concentration of PT mutant determined by ELISA expressed as apercentage. ADPR activity is the extent of ADP-ribosylation of bovinetransducin catalysed by a PT analogue, relative to that catalysed by anequal concentration of wild-type PT, expressed as a percentage. S1epitope refers to the expression of an immunodominant S1 epitoperecognized by a specific monoclonal antibody PS21 (ATCC HB 10299deposited Nov. 30, 1989), as compared with the wild-type PT (+++++). NDdenotes not determined.

TABLE 2 Functional amino acid residues in pertussis toxin f r mutationSubunit Residues Preferred Replacement S1 Phe-23 Asp or Glu Ser-48 AlaVal-51 Ile Gln-127 Ala or Asp Leu-131 Lys or Arg Gly-199 Val or GlnAla-200 Ile Phe-235 Glu S2 His-15 Ala or Thr Gln-16 Ala or Thr Trp-52Val Glu-66 Ala or Lys Asp-81 Ala or Ser Leu-82 Ala or Glu Lys-83 GluSer-104 Ala Arg-125 Ala Ser-147 Thr Arg-150 Ser Lys-151 Ser S3 Gln-15Ala or Thr Gln-16 Ala or Thr Tyr-82 Ala or Val Arg-83 Glu Ser-104 AlaArg-125 Ala Arg-150 Ser Arg-151 Ser S4 Asp-1 Ala Tyr-4 Ala or Val Gly-60Val Ser-61 Ala Glu-65 Ala Arg-69 Ala Thr-88 Val Pro-93 Ala Asp-54 GluThr-51 Tyr Thr-55 Tyr Gly-58 Val S5 Ser-62 Ala

TABLE 3 Immunogenicity of the Pertussis toxin component of a DTPacellular vaccine and an acellular DTP vaccine containing a geneticallydetoxified PT analog Reciprocal Anti-pertussis toxin IgG CHO cellVaccine Formulation Reactive titre neutralization titre DTP acellularvaccine 1,363,678 50.8 Recombinant acellular 1,363,678 40.3 vaccine

REFERENCES

-   1. Bergstrand H., Andersson I., Nystrom I., Pauwels R.,    Bazin H. (1983) The Non-specific enhancement of allergy. II.    Precipitation of anaphylactic in vitro response capacity and serum    IgE and IgG2a antibody synthesis in primed but non-responding rats    by injection of alum. Allergy 38:247–260-   2. Cogne M., Ballet J. J. Schmitt C., Bizzini B. (1985) Total and    IgE antibody levels following booster immunization with aluminum    adsorbed and nonadsorbed tetanus toxoid in humans. Ann. Allergy    54:148–151-   3. Nagel J., Svec D., Waters T., Fireman P. (1977) IgE Synthesis in    Man: I. Development of specific IgE antibodies after immunization    with Tetanus-Diphtheria (Td) toxoids. J. Immunol. 118:334–341-   4. Hedenskog S., Bjorksten B., Blennow M., Granstrom G.,    Granstrom M. (1989) Immunoglobulin E response to pertussis toxin in    whooping cough and after immunization with a whole cell and an    acellular pertussis vaccine. Int. Arch. Allergy Appl. Immunol.    89:156–161.-   5. World Health Statistics (1992) immunization coverage. World    Health Organization, Geneva, pp. 19–24.-   6. Medical Research Council (1951) Br. Med. J. 2:1464–1472.-   7. Medical Research Council (1956) Br. Med. J. 2:454–462.-   8. Medical Research Council (1959) Br. Med. J. 1:994–1000.-   9. Fine P. E., Clarkson J. A. (1987) Reflections on the efficacy of    pertussis vaccines. Rev. Infect. Dis. 9:866–883.-   10. Kanai K. (1980) Japan's experience in pertussis epidemiology and    vaccination in the past thirty years. Jpn. J. Med. Sci. Biol.    33:107–143.-   11. Miller D. L. Alderslade R., Ross E. M. (1982) Whooping cough and    whooping cough vaccine: the risks and benefits debate. Epidemiol.    Rev. 4:1–24.-   12. Romanus V., Jonsell R., Bergquist S. O. (1988) Pertussis in    Sweden after the cessation of general immunization in 1979. Pediatr.    Infect. dis. 6:364–371.-   13. Munoz J. J., Arai H., Bergman K., Sadowski P. L. (1981)    Biological activities of crystallin pertussigen from Bordetella    pertussis. Infect. Immun. 33:820–826-   14. Marwick C. (1988) Pertussis vaccines: Trials and Tribulations.    JAMA 259:2057–2059.-   15. Storsaeter J., Hallander H., Farrington C. P., Olin P., Molby    R., Miller E. (1990) Secondary analyses of the efficacy of two    acellular pertussis vaccines evaluated in a Swedish phase III trial.    Vaccine 8:457–461.-   16. Loosmore, S. Zealey, G., Cockle S. Boux, H., Chong, P.,    Yacoob, R. and Klein, M. (1993) Characterization of pertussis toxin    analogs containing mutations in B-oligomer subunits. Infect. Immun.    61:2316–2324.-   17. Burnette W. N., Cieplak W., Smith S. G., Keith J. M. (1989)    Effects of mutations on enzyme activity and immunoreactivity of the    S1 subunit of pertussis toxin. Infect. Immun. 57:3660–3662.-   18. Loosmore S., Cockle S., Zealey G., Boux H., Cockle S., Radika    K., Fahim R., Zobrist G., Yacoob R. K., Chong P., Yao F. L.,    Klein M. (1990) Engineering of genetically detoxified pertussis    toxin analogs for development of a recombinant whooping cough    vaccine. Infect. Immun. 58:3653–3662.-   19. Nencioni L., Pizza M., Bugnoli M., DeMagistris T., Di Tomasso    A., Giovannoni F., Manetti R., Marsili I., Matteucci G., Nucci D.,    Olivieri R., Pileri P., Presentini R., Villa L., Kreeftenberg J. G.,    Silvestri S., Tagliaube A., Rappuoli R. (1990) Characterization of    generically inactivated pertussis toxin mutants: Candidates for a    new vaccine against whooping cough. Infect. Immun. 58:1308–1315.-   20. Lobet Y., Cieplak W., Mar V. L., Kaljot K. T., Sato H.,    Keith J. M. (1988) Pertussis toxin S1 mutant with reduced enzyme    activity and a conserved protective epitope. Science 242:72–74.-   21. Pizza M., Covacci A., Bartolini A., Perugini M., Nencioni L.,    DeMagistris T., Villa L., Nucci D., Manetti R., Bugnoli M.,    Giovannoni F., Olivieri R., Barbieri J., Sato H., Rappuoli R. (1989)    Mutants of pertussis toxin suitable for vaccine development. Science    246:497–499.-   22. Podda A., Nencioni L., Demagistris M., Di Tomasso A., Bossu P.,    Nuti S., Pileri P., Peppoloni S., Bugnoli M., Ruggiero P., Marsili    I., D'Errico A., Tagliabue A., Rappuoli R. (1990) Metabolic, humoral    and cellular responses in adult volunteers immunized with the    genetically inactivated pertussis toxin mutant PT-9K/129G. J. Exp.    Med. 172:861–868.-   23. Mills K. H. G., Barnard A., Watkins J., Redhead K. (1993)    Cell-mediated immunity to Bordetella pertussis: Role of Th1 cells in    bacterial clearance in a murine respiratory infection model. Infect.    Immun. 61:399–410.-   24. Redhead K., Watkins J., Barnard A., Mills. K. H. G. (1993)    Effective immunization against Bordetella pertussis respiratory    infection in mice is dependent on induction of cell-mediated    immunity. Infect. Immun. 61:3190–3198.-   25. De Magistris M., Romano M., Nuti S., Rappuoli R.,    Tagliabue A. (1988) Dissecting human T cell responses against    Bordetella species. J. Exp. Med. 168:1351–1362.-   26. Gearing A. J. H., Bird C. R., Redhead K., Thomas M. (1989) Human    cellular immune responses to Bordetella pertussis infection. FEMS    Microbiol. Immunol. 47:205–212.-   27. Tomoda T., Ogura H., Kurashige T. (1991) Immune responses to    Bordetella pertussis infection and vaccination. J. Inf. Dis.    163:559–563.-   28. Petersen J. W., Ibsen P. H., Bentzon M. W., Capiau C.,    Heron I. (1991) The cell mediated and humoral immune response to    vaccination with acellular and whole cell pertussis vaccine in adult    humans. FEMS Microbial. Immunol. 76:279–288.-   29. Podda A., DeLuca E., Titone L., Casadel A., Cascio A., Peppoloni    S., Volpini G., Marsili I., Nencioni L., Rappuoli R. (1992)    Acellular pertussis vaccine composed of genetically inactivated    pertussis toxin: Safety and immunogenicity in 12-to-24 and 2-to-4    month old children. J. Pediatr. 120:680–685.-   30. Podda A., Nencioni L., Marsili I., Peppoloni S., Volpini G.,    Donati D., Di Tommaso A., De Magistris T., Rappuoli R. (1991) Phase    I clinical trial of an acellular pertussis vaccine composed of    genetically detoxified pertussis toxin combined with FHA and 69 RD.    Vaccine 9:741–745.-   31. Nencioni L., Volpini G., Peppoloni S., Bugnoli M., DeMagistris    T., Marsili I., Rappuoli R. (1990) Properties of Pertussis toxin    mutant PT-9K/129G after formaldehyde treatment. Infect. Immun.    59:625–630.-   32. Marsili I., Pizza M., Giovannoni F., Volpini G., Bartalini M.,    Olivieri R., Rappuoli R., Nencioni L. (1992) Cellular pertussis    vaccine containing a Bordetella pertussis strain that produces a    nontoxic pertussis toxin molecule. Infect. Immun. 60:1150–1155-   33. Long S. S., Deforest A., Pennridge Pediatric Associates,    Smith D. G., Lazaro C., Wassilak G. F. (1990) Longitudinal study of    adverse reactions following Diphtheria-Tetanus-Pertussis vaccine in    infancy. Pediatrics 85:294–302.-   34. Butler N. R., Voyce M. A., Burland W. L., Hilton M. J.    Advantages of aluminum hydroxide adsorbed combined diphtheria,    tetanus, and pertussis vaccines for the immunization of infants. Br.    Med. J. 1:663–666.-   35. Aprile M. A., Wardlaw A. C. (1966) Aluminum compounds as    adjuvants for vaccines and toxoids in man: A review. Can J. Pub.    Health 57:343–354.-   36. Pineau A., Durand C., Guillard O., Bureau B., Stalder J. (1992)    Role of aluminum in skin reactions after    diphtheria-tetanus-pertussis-poliomyelitis vaccination: An    experimental study in rabbits. Toxicology 73:117–125.-   37. Goto N., Akama K. (1982) Histopathological studies of reactions    in mice injected with aluminum-adsorbed tetanus toxoid. Microbiol.    Immunol. 26:1121–1132.-   38. Erdohazi M., Newman R. L. (1971) Aluminum hydroxide granuloma.    Br. Med. J. 3:621–623-   39. Bernier R. H., Frank J. A., Nolan T. F. (1981) Abscesses    complicating DTP vaccination. Am. J. Dis. Child. 135:826–828-   40. Cox N. H., Moss C., Forsyth A. (1988) Cutaneous reactions to    aluminum in vaccines: an avoidable problem. Lancet ii, 43.-   41. Strom J. (1967) Further experience of reactions, especially of a    cerebral nature, in conjunction with triple vaccination: A study    based on vaccinations in Sweden 1959–1965. Br. Med. J. 4:320–323.-   42. Lione A. (1986) More on aluminum in infants. New England J. Med.    314:923-   43. Gupta R. K. 7 Sharma S. B., Ahuja S., Saxena S. N. (1987) The    effect of aluminum phosphate adjuvant on the potency of pertussis    vaccine. J. Biol. Stand. 15:99–101.-   44. Sar so J. S., Bahrawi W., Witjaksono 14, . . . , Budiarso R. L.    P., Brotowasisto B., Dewitt W. R., Gomez C. Z. (1978) A controlled    field trial of plain and aluminum hydroxide adsorbed cholera    vaccines in Surabaya, Indonesia, during 1973–1975. Bull. WHO 56:619.-   45. Collier L. H., Polakoff S., Mortimer J. (1979) Reactions and    antibody responses to reinforcing doses of adsorbed and plain    tetanus vaccines. Lanct i:1364.-   46. Gupta R. K., Relyveld E. H. (1991) Adverse reactions after    injection of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine are    not due only to pertussis organisms or pertussis components in the    vaccine. Vaccine 9:699–702.-   47. Granstrom M., Granstrom P., Gillenius P., Askelof P. (1985)    Neutralizing antibodies to pertussis toxin in whooping cough. J.    Infect. Dis. 151:646–649.-   48. Mosmann T. R., Schumacher J. H., Street N. F., Budd R., O'Garia    A., Fong T., Mond M. W., Moore W. M., Sner A.,    Fiorentino, D. F. (1991) Diversity of cytokine synthesis and    function of mouse CO4 T-cells. Imm. Rev. 123:219–229/-   49. Mosmann T. R., Cherwinski H., Bond H. W., Gredlin A.,    Coffman R. L. (1986) Two types of murine helper T cell clone, I.    Definition according to profiles of cytokine activates and secreted    proteins. J. Immunol. 136: 2348–2357.-   50. Mosmann T. R., Coffman R. L. (1989) T_(h)1 and T_(h)2 cells:    Different patterns of lymphokine secretion lead to different    functional properties. Ann. Rev. Immunol. 7:145–173.-   51. Coffman R. L., Seymour B. W. P., Debman D. A., Hivaki D. D.,    Christiansen J. A., Shrader B. chervinski H. M., Savelkoul H. P. J.,    Finkelman F. D., Bond M. W., Mosmann T. R. (1988) The role of helper    T cell products in mouse B-cell differentiation and isotype    regulation. Immunol. Rev. 102:5–28.-   52. Romagnani S. (1991) Human T_(h)1 and T_(h)2 subsets: Doubt no    More. Immunol. Today 12: 256–257.-   53. Coffman R. L., Varkila K., Scott P., Chatelain R. (1991) Role of    cytokines in the differentiation of CD4 T cell subsets in vno. Imm.    Rev. 123:189–207.-   54. Bystryn J-C, Bart R. S., Livingston P., Kopf A. W. (1974) Growth    and immunogenicity of murine B-16 melanoma. Journal of Investigative    Dermatology, 63, 369–373.

1. An immunogenic composition comprising: a geneticallydetoxified-pertussis holotoxin, and at least one, non-Bordetella antigencomprising inactivated tumour cells or membrane fraction thereof whereinsaid genetically detoxified-pertussis holotoxin is present in an amountsufficient to modulate an immune response to said non-Bordetella antigenin the absence of extrinsic adjuvant.
 2. The composition of claim 1wherein said cells are inactivated by irradiation.
 3. The composition ofclaim 1 wherein at least one amino acid is removed or replaced in saidgenetically detoxified-pertussis holotoxin.
 4. The composition of claim3 wherein multiple amino acids are removed or replaced in saidgenetically detoxified-pertussis holotoxin.
 5. The composition of claim3 or 4 wherein said at least one amino acid is selected from the groupconsisting of (SI) ARG9, ARG13, TRP26, ARG58, and GLU129.
 6. Thecomposition of claim 4 wherein said multiple amino acids are (SI) ARG9,GLU129.
 7. The composition of claim 6 wherein said multiple amino acidsare (SI) ARG9 to LYS9 and GLU129 to GLY129.
 8. A method of obtaining amodulated immune response to an antigen in a host comprising:administering at least one, non-Bordetella antigen to said host, saidantigen comprising inactivated tumour cells or a membrane fractionthereof, and a genetically detoxified pertussis holotoxin in an amountsufficient to modulate an immune response to said non-Bordetella antigenin the absence of extrinsic adjuvant.
 9. The method of claim 8 whereinsaid host is a human.
 10. The method of claim 8 wherein said cells areinactivated by irradiation.